Cyanin-type compounds having an alkynyl linker arm

ABSTRACT

Cyanine-type fluorescent dyes modified with an alkynyl linker arm of formula (I), suitable for the conjugation of biomolecules, such as for example nucleosides, nucleotides, oligonucleotides, nucleic acids, proteins, peptides, vitamins and hormones. A method and intermediates for the synthesis of the alkynyl cyanines of the invention are also described, as well as alkynyl cyanine-biomolecule conjugates and methods for preparing thereof. The alkynyl cyanines can be advantageously used as markers for biomolecules or as quenchers.

The present invention concerns cyanine type fluorescent dyes having analkynyl linker arm, their synthesis and their use in bioconjugation andfluorescent labelling of biomolecules such as, e.g., nucleosides,nucleotides, nucleic acids (DNA, RNA or PNA) and proteins, as well astheir use in the synthesis of large “Stokes shift” fluorescent dyes.

Furthermore in some embodiments the cyanines of the invention aresuitable for use in the double labelling of both nucleic acids andproteins or other biomolecules. In other embodiments the cyanines of theinvention are suitable for use as quenchers, i.e., molecules capable ofquenching the fluorescence emitted by fluorophores, to be used instructures of the type “molecular beacons”.

The use of fluorescence technology has become widespread in the areas ofmolecular biology, genomics, proteomics and analytical chemistry, sinceit enables very sensitive and specific test to be carried out, competingeffectively with radiolabelling and enzymatic labelling techniques.

DNA probes labelled with fluorescent dyes are valuable reagents for theanalysis and separation of molecules. Specific applications of suchfluorescent probes include:

-   -   1. automated DNA sequencing and mapping;    -   2. determination of the concentration of a substance that binds        to a second species, e.g. DNA hybridisation reactions, in        techniques like real time PCR and molecular recognition via        molecular beacons;    -   3. localization of biomolecules in cells, tissues or insoluble        supports by techniques such as fluorescence staining.

Also protein labelled with fluorescent substances are very powerfulanalytical tools used in techniques such as fluorescence microscopy,fluorescence immunoassays, protein chips, laser induced fluorescencecapillary electrophoresis.

Recently, techniques making use of probes containing marker pairs, oneof which is a fluorescence emitter and the other is a quencher,established themselves for the detection of nucleic acids. The twomolecules are, e.g., conjugated to the ends of an oligonucleotidic probehaving a central target nucleic acid binding sequence. Theoligonucleotide probe takes on a closed loop conformation by virtue ofthe presence of two short complementary base sequences (generally 5 to10 bases each) flanking the central target binding sequence. In theabsence of the target the two flanking sequences are capable oforiginating an intramolecular base pairing hybridized structure, inwhich the quencher is proxymal to the fluorophore, thereby quenching itsfluorescence. When the oligonucleotide probe finds a target sequencecomplementary to its own central binding sequence, it unfolds tohybridize with it, and the fluorophore and the quencher space out sothat the quencher is no longer able to quench the fluorescence emittedby the fluorophore, thus allowing the detection of the emittedfluorescence in the presence of the target.

Among fluorescent dyes, cyanines have wide application as biomoleculelabels in several bioanalytical techniques thanks to theirchemical-physical properties such as the high extinction coefficient,high quantum yield, independence of pH, low molecular weight and thepossibility to perform multiple assays simultaneously using multiplefluorophores emitting at different wavelengths. The cyanines can be alsoused as quenchers if their structure contains, for instance, nitrogroups.

To be useful as a fluorescent label or as a quencher forbioconjugations, a dye has to be provided with a suitable linker armcontaining a functional group to give rise to a covalent link with thebiomolecule which is to be labelled. While the chromogenic part of thedye structure is generally known, the introduction of a functionalizedlinker arm for the purpose of conjugation with another molecule,represents the innovative step in a number of inventions concerning theuse of fluorescent dyes as labelling reagents. The research in thisfield is therefore focused on innovative functionalized arms, since thechemistry and the behaviour of such functionalized arms can remarkablyaffect the fluorescence of the whole molecule as well as severalphysical and chemical characteristics such ashydrophobicity/hydrophilicity, stacking and intramolecular quenching.

In general, the presence of only one functionalized linker arm ispreferable, in order to avoid cross-linking between multiple similarmolecules, undesired multiple reactions or purification problems.

The synthesis of biomolecules labelled with fluorescent compounds, e.g.nucleotides, requires several steps among which the separate synthesisof a functionalized nucleotide and of a functionalized fluorescentmolecule and their subsequent conjugation.

Several kinds of functionalized linker arms suitable for the conjugationof biomolecules with fluorescent compounds are described in the priorart.

For instance U.S. Pat. No. 5,486,616 describes a method for labellingbiomolecules with water soluble cyanines containing a functionalizedlinker arm consisting of an alkyl chain terminating with a functionalgroup selected e.g. among isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono- or di-halogen substitutedpyridine, mono- or di-halogen substituted diazine, maleimide, aziridine,sulphonyl chloride, acyl chloride, hydroxysuccinimidyl ester,hydroxysulfosuccinimidyl ester, imidic ester, hydrazine,azidonitrophenyl, azide, 3-(2-pyridyldithio)-propionamide, glyoxal,aldehyde.

Furthermore, U.S. Pat. No. 5,047,519 discloses a method for preparingfluorescently labelled nucleotides comprising the steps of:

-   -   activating the nucleotide towards aromatic nucleophilic        substitution by means of the introduction of a iodine atom at        the 5 position of a pyrimidine, at the 8 position of a purine or        at the 7 position of a 7-deazapurine;    -   phosphorilation of the iodonucleotide in order to obtain the        corresponding triphosphate;    -   introducing a propargylamino functional group at the activated        position by means of a nucleophilic attack following the Heck        Pd(0) catalyzed reaction;    -   synthesizing a fluorescent dye containing a carboxylic acid;    -   preparing the fluorescent dye active ester in order to activate        it towards the acyl nucleophilic substitution reaction;    -   reacting the propargylamino modified nucleotide with the        fluorescent dye active ester.

Alternatively, the functionalized linker arm may contain an acid group,which must be then activated to react with an amine group of the dyemolecule to give an amide bond.

Anyway in both cases the activation of the carboxylic acid group, e.g.as active ester, is necessary.

Nevertheless, the use of active esters, and in general of activecarboxylic groups, shows considerable disadvantages such as poorstability over time and difficult synthesis. Active esters are, in fact,not much stable molecules if not in perfectly anhydrous conditions andtherefore suffer from substantial storage troubles. They tend to degradeover time by hydrolysis and the percentage of active product in apackage decreases over time. Furthermore, due to the poor stability, itis virtually impossible to store the unused product still furtherimmediately after opening of the packaging. Moreover, the need to workin perfectly anhydrous conditions makes the synthesis of such compoundsdifficult and expensive, since the purifications are to be carried outwith anhydrous solvents.

An object of the present invention is to provide a fluorescent dyemolecule of the type cyanine provided with a linker arm suitable for theconjugation with a biomolecule, such as for example nucleosides,nucleotides, nucleic acids and proteins, which however does not requirethe formation on an active acid group during the conjugation reaction.

Such object is achieved by a cyanine modified with an alkynyl-linkerarm, having the following general formula (I), including the valencetautomers thereof:

wherein

R₁ is a linear, saturated or unsaturated alkyl chain, having from 1 to30 carbon atoms, wherein one or more carbon atoms are each optionallyreplaced with a component independently selected from an oxygen or asulfur atoms, a —NH— or a —CONH— group, or a cyclic, aromatic or notaromatic, 4-, 5- or 6-membered grouping of carbon atoms wherein one ormore carbon atoms are each optionally replaced with a heteroatomindependently selected from oxygen, sulfur, nitrogen and selenium; W₁and W₂ are independently selected from a benzene ring and a naphthalenering wherein one or more carbon atoms are optionally replaced with oneor more heteroatoms selected from oxygen, sulfur, selenium and nitrogen,or one of W₁ and W₂ is absent, or both of them are absent; X₁ and X₂ areindependently selected from the group consisting of —O—, —S—, —Se—, —N—,—C(CH₃)₂, —CH═CH—, —NH—, or

with j=1-20 and k=1-20;

R₂, R₃, R₄, R₅ and R₆ are indipendently selected from hydrogen, —COOH,—OH, —NO₂, —OCH₃, —O₃H, —O₃, and —R₈—Y wherein R₈ is a linear, saturatedor unsaturated alkyl chain, having from 1 to 30 carbon atoms, whereinone or more carbon atoms are each optionally replaced with a componentindependently selected from an oxygen or a sulfur atom, a —NH— or a—CONH— group, or a cyclic, aromatic or not aromatic, 4-, 5- or6-membered grouping of carbon atoms wherein one or more carbon atoms areeach optionally substituted by a heteroatom independently selected fromoxygen, sulfur, nitrogen or selenium, and wherein Y is selected from thegroup consisting of hydrogen, carboxyl, carbonyl, amino, sulphydryl,thiocyanate, isotyocianate, isocyanate, maleimide, hydroxyl,phosphoramidite, glycidyl, imidazolyl, carbamoyl, anhydride,bromoacetamido, chloroacetamido, iodoacetamido, sulfonyl halide, acylhalide, aryl halide, hydrazide, succinimidyl ester,hydroxysulfosuccinimidyl ester, phthalimidyl ester, naphthalimidylester, monochlorotriazine, dichlorotriazine, mono- or di-halidesubstituted pyridine, mono- or di-halide substituted diazine, aziridine,imidic ester, hydrazine, azidonitrophenyl, azide,3-(2-pyridyldithio)-propionamide, glyoxal, aldehyde, nitrophenyl,dinitrophenyl, trinitrophenyl and —C≡CH;

M is a counterion; and

Q is a polymethinic chain selected from:

wherein R₇ is selected from the group consisting of hydrogen, fluorine,chlorine, bromine, iodine, phenoxy, thiophenoxy, anilino,cyclohexylarnino, pyridine, —R₈—Y, —O—R₈—Y, —S—R₈—Y, —NH—R₈—Y, whereinR₈ e Y are as defined above, and aryl optionally substituted with one ormore substituents independently selected from the group consisting of—SO₃H, carboxyl (—COOH), amino (—NH₂), carbonyl (—CHO), thiocyanate(—SCN), isothiocyanate (—CNS), epoxy and —COZ wherein Z represents aleaving group.

Suitable leaving groups are for example —Cl, —Br, —I, —OH, 13 OR₁₁,—OCOR₁₁, wherein R₁₁ is linear or branched lower C₁-C₄ alkyl (forexample methyl, ethyl, t-butyl or isopropyl), —O—CO—Ar, wherein Ar isoptionally substituted aryl; —O—CO—Het, wherein Het is selected fromsuccinimide, sulfosuccinimide, phthalimide and naphthalimide, —NR₂₂R₃₃,wherein R₂₂ and R₃₃ are each independently linear or branched C₁-C₁₀alkyl.

As used above, the expression “carbon atom optionally replaced with”means that such carbon atom in the linear alkyl chain or in the cyclicgrouping of atoms can be replaced by one of the components orheteroatoms indicated above.

In the following of the description, the cyanines having an alkynyllinker arm of the present invention illustrated by formula (I) will bereferred to as “alkynyl cyanines”.

Preferred examples of alkynyl cyanines which fall within the scope ofthe present invention are:

wherein Q and R₈ are as defined above for formula (I) an n is an integercomprised between 1 and 100.

The alkynyl cyanines of the present invention are synthetized accordingto a reaction scheme which comprises the following steps:

-   -   1. synthesis of the quaternary ammonium salt    -   2. synthesis of a second quaternary ammonium salt    -   3. synthesis of the hemicyanine    -   4. synthesis of the cyanine

Step 1 is carried out by reacting in a suitable solvent, such assulfolane, acetonitrile or N,N-dimethylformamide, a nitrogen-containingheterocyclic system with a molecule which contains a terminal triplebond to form the alkynyl quaternary ammonium salt:

wherein X₂, R₁, R₄, R₆ e W₂ are as defined above for formula (1), and R₉is selected from the group consisting of iodine, chlorine, bromine, OH,sulphate and tosylate.

Step 2 consists of the synthesis of a second quaternary ammonium saltstarting from a second nitrogen-containing heterocyclic system and froman alkylating molecule R₂—R₉ according to the scheme:

wherein X₁, R₂, R₃, R₅ e W₁ are as defined above for formula (I), and R₉is selected from the group consisting of iodine, chlorine, bromine, OH,sulphate, tosylate.

Step 3 can be carried out either on the alkynyl-quaternary ammonium saltA of step 1 or on the quaternary ammonium salt B synthetized in step 2.It consists of the reaction of A or B with a compound capable ofreacting with the heterocyclic quaternary ammonium salt to form apolymethine chain. Non limiting examples of such compounds aretriethylorthoformate, N,N-diphenylformamide, malonaldehyde dianil,pyridyl malonaldehyde, trimethoxypropene,5-phenylamino-2,2-trimethylene-2,4-pentadienylidene phenylammoniumchloride, chloromalonaldehyde dianil and squaric acid. From this step,an intermediate named hemicyanine is obtained.

Step 4 is carried out by reacting the hemicyanine obtained in step 3with the quaternary ammonium salt A or B not used in the preceding step.The desired alkynyl cyanine product is obtained.

In all of the preceding steps the specific reaction conditions depend onthe type of the reagents employed in the different steps and on thedesired final product.

The alkynyl cyanines of the present invention are particularly suitablefor the conjugation of biomolecules, particularly proteins, nucleosides,nucleotides, oligonucleotides and in general with nucleic acidscontaining bases modified with an halogen atom (for example iodine,chlorine, bromine) at position 5 of pyrimidines, 8 of purines and 7 of7-deazapurines.

Thus, the scope of the present invention also encompasses an alkynylcyanine as previously described conjugated through the R₁—C≡CH linkerarm with a biomolecule selected from the group consisting ofnucleosides, nucleotides, oligonucleotides, nucleic acids, for exampleDNA, RNA or PNA (“peptide nucleic acids”), peptides and proteins.

Such a cyanine conjugated with a biomolecule can be represented by thefollowing general formula (II):

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, X₁, X₂, W₁, W₂, M and Q are asdefined for formula (I).

With reference to nucleosides, the general conjugation scheme is asfollows:

wherein B is a base selected from cytosine, uracil, guanine, adenine,xanthine, hipoxanthine, 7-deazaguanine, 7-deazaadenine, 7-deazaxanthine,7-deazaipoxanthine, Alg is an halogen selected from iodine, chlorine andbromine, and wherein R₁ is as defined in formula (I).

In prevoius Scheme 1, the the alkynyl arm-containing cyanine accordingto the invention is reacted with the halo-derivative of a nucleoside.Halo-derivatives of nucleotides and nucleosides are commerciallyavailable or can be synthetized as described in European patentEP0251786B1.

The reaction is carried out in a suitable anhydrous organic solvent, forexample anhydrous N,N-dimethylformamide (DMF), and in the presence of anorganic base, such as for example triethylamine (N(C₂H₅)₃), and of aPd(0) compound as the catalyst. Instead of the Pd(0) compound, it ispossible to use as the catalyst a Pd(II) compound (for example(C₆H₅)₂PdCl₂)) and a co-catalyst capable of forming Pd(0) in situ, suchas for example CuI.

The reaction is preferably carried out at room temperature for a periodof time of about 8 hours. When the reaction time has passed, a secondaliquot of catalyst, organic base and optionally co-catalyst is added tothe reaction mixture, then the mixture is left reacting under the sameconditions for a further period of time, preferably about 16 hours.

Reaction Scheme 1 also applies to the conjugation of nucleotides,oligonucleotides and nucleic acids (DNA, RNA, PNA).

Alternatively, the cyanine-nucleoside conjugate obtained according toScheme 1 can be phosphorylated, thereby obtaining a cyanine-nucleotidetriphosphate conjugate. Phosphorylation of the cyanine-nucleosideconjugate can be carried out for example using the nucleosidephosphorylation method described in patent EP0251786B1.

The cyanine-nucleotide triphosphate conjugate is a suitable substratefor DNA polymerase and may be used in PCR reactions for the preparationof fluorescent DNA chains.

The scope of the present invention also encompasses an alkynyl cyanineas previously described, conjugated through the R₁—C≡CH linker arm witha second fluorescent dye, the second fluorescent dye being capable ofemitting fluorescence at a wavelength at which the cyanine is capable ofabsorbing, or the second fluorescent dye being capable of absorbing at awavelength at which the alkynyl cyanine is capable of emitting (FRETcouples).

Said cyanine conjugated with a second fluorescent dye can be representedby the following general formula (III):

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, X₁, X₂, W₁, W₂, M and Q are asdefined above for formula (I) and DYE is the second dye.

Such conjugates are useful when it is desired to have a fluorescentsystem with a large Stokes shift in order to have maximum separationbetween excitation light and emission.

Optical bioanalytical strumentation in fact uses filters in order tooptimally separate excitation light from emission, but in the case offluorophores with a small Stokes shift overlappings may occur which makefluorescence quantification difficult.

One example of a conjugate between a cyanine and a second fluorescentdye forming a large Stokes shift is the compound1-(4-carboxybutyl)-1′-{6-[N,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrryl]-es-5-inyl}-3,3,3′,3′-tetramethyl-5,5′-disulfoindodicarbocyanine sodium salt of formula:

which is obtained by reacting1-(4-carboxybutyl)-1′-es-5-inyl-3,3,3′,3′-tetramethyl-5,5′-disulphonateindodicarbocyanine sodium salt withN,N′-difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipirryne according toa mechanism which is similar to the one described for the conjugation ofan alkynyl cyanine with an halo-nucleoside.

Other examples are conjugates obtained by reacting alkynyl cyanines ofthe general formula (I) with complexes of transition metals (Me) such asruthenium, osmium, rhenium, iridium, rhodium, platinum, palladium,molibdenum and technetium, having at least a nitrogen-containingheterocyclic linker linked to the cyanine alkynyl linker arm.Nitrogen-containing heterocyclic linkers suitable for this purpose arefor example optionally substituted 2,2′-bipiridyne, optionallysubstituted 1,10-phenantroline, optionally substitutedbatophenantroline. Suitable substituents for the above mentionednitrogen-containing heterocyclic linkers are for example iodine,chlorine, bromine, phenyl, trifluoromethyl, or the R₅ group defined informula (I).

A specific non-limiting example of such a conjugate is the compound1-(4-carboxybutyl)-1′-{6-[(2-2′-bipyridine)₂(2-2′bipyridin4-yl)ruthenium(II)]-es-5-inyl}-3,3,3′,3′-tetramethyl-5,5′-disulphonateindodicarbocyanine iodide:

Such a compound is synthetized by reacting the alkynyl cyanine accordingto the present invention1-(4-carboxybutyl)-1′es-5-inyl-3,3,3′,3′-tetramethyl-5,5′-disulphonateindodicarbocyanine sodium salt with the compound bis(2,2′-bipyridine)(4-iodo-2,2′,bipyridine) ruthenium(II) with a procedure similar to theone of Scheme 1 in which the halo-nucleoside is replaced by theruthenium complex.

A compound of this type has very interesting optical properties whichmake it particularly useful as a label in bioanalytical applications.

Further preferred embodiments of the present invention are the alkynylcyanines illustrated by formula (I) in which one of R₂, R₃, R₄, R₅ andR₆ is —R₈—Y wherein Y is different from H and from —C≡CH. Such cyaninesin fact contain a second functional group which is capable of binding toa second biomolecule, for example a protein or a peptide.

Cyanines according to said preferred embodiment are bifunctionalmolecules, thereby permitting the simultaneous conjugation of twodifferent or similar biomolecules.

Therefore, the scope of the present invention also encompasses abifunctional alkynyl cyanine conjugated through the —R₁—C≡CH linker armwith a first biomolecule selected from the group consisting ofnucleotides, nucleosides, nucleic acids, proteins, peptides, vitaminsand hormones, and through the —R₈—Y linker arm with a second similar ordifferent biomolecule selected from the group consisting of nucleotides,nucleosides, nucleic acids, proteins, peptides, vitamins and hormones.

Such an alkynyl cyanine conjugated with a first and a second biomoleculeis represented by the following general formula (IV):

wherein R₁, R₃, R₄, R₅, R₆, R₇, R₉, X₁, X₂, W₁, W₂, M and Q are asdefined for formula (I).

Bifunctional alkynyl cyanines of the invention may be used for examplefor preparing fluorescent oligonucleotides conjugated with the proteinstreptavidin, useful for use in molecular recognition assays with signalenhancement. Streptavidin in fact can bind four biotin molecules withhigh affinity. If every biotin is in turn conjugated with a fluorescentcyanine, the total detectable fluorescent signal will be 5-folds higher,with a remarkable increase in the assay sensitivity. The followingscheme shows the structure of the conjugate described above:

The following examples, which illustrate the synthesis of someembodiments of the present invention, are provided for illustrationpurposes only and should not be intended to limit the scope of theinvention in any way.

EXAMPLE 1 Synthesis of1-ethyl-1′-(hex-5-ynyl)-3,3,3′,3′-tetramethyl-5,5′-disulfo-indomonocarbocyaninepotassium salt (Cyanine IRIS 3 sulfo alkynyl) (Compound 1) a) Synthesisof 6-iodo-hex-1-yne

A mixture of 6-chlorohex-1-yne (10 g, 85.8 mmol ) and 71 ml of anhydrousacetone is heated at 70° C. on a water bath for 10 min. in around-bottom flask fitted with a reflux condenser closed by a calciumchloride trap. Sodium iodide (25.8 g, 172 mmol) is added to the mixtureand heating is maintained for 22-24 h. The mixture is then cooled toroom temperature and concentrated by distillation. Ether is added andthe inorganic salt, which precipitates, is filtered. The residue ispoured in a separatory funnel, which is then shaken, successively, with10% sodium bisulfite sodium bicarbonate solution. It is dried withanhydrous sodium sulfate and the solvent is evaporated under reducedpressure.

b) Synthesis of N-hex-5-ynyl-2,3,3-trimethylindoleninium-5-sulfonate

A mixture of 2,3,3-trimethyl-3H-indolenine-5-sulfonate potassium salt(5.0 g, 18.02 mmol), 6-iodohex-1-yne (22.56 g, 180.2 mmol) and 50 ml ofsulfolane is placed in a round-bottom flask fitted with a refluxcondenser and refluxed at 120° C. on a water bath for 12 h. The reactionmixture is cooled to room temperature and and added dropwise to 800 mlof vigorous stirred diethyl ether. The product is collected on a frittedglass filter, washed with ether and dried in a desiccator under vacuum.

c) Synthesis of2-{(E)-2-[acetyl(phenyl)amino]vinyl}-1-ethyl-3,3-dimethyl-3H-indolium-5-sulfonate(hemicyanine)

3.00 g of N-ethyl-2,3,3-trimethylindoleniniun-5-sulfonate (8.43 mmol),3.33 g of N,N-diphenylformamide (16.98 mmol), 6.00 ml ofacethylchloride, 60.00 ml of acetic anhydride are placed in a 250 mlround-bottom flask. The mixture is heated at 120° C. for 90 minutes. Theorange solution is cooled to room temperature and added dropwise to 300ml of vigorous stirred diethyl ether. The product is collected on afritted glass filter, washed with diethyl ether and dried in adesiccator under vacuum. The desidered product has an absorbance maximumat 378 nm in methanol. Yield is 97%.

d) Synthesis of cyanine IRIS 3 sulfo alkynyl

5.00 g (12.14 mmol) of the hemicyanine synthesized in the previous stepis placed in a 250 ml round-bottom flask together with 3.87 g (12.14mmol) of N-hex-5-ynyl-2,3,3-trimethylindoleninium-5-sulfonate, 10.80 mlof triethylamine and 109 ml of acetic anhydride and heated at 135° C.for 2 h. The red purple solution is cooled to room temperature and addeddropwise to 800 ml of vigorous stirred diethyl ether. The desiredproduct is collected on a fritted glass filter, washed with diethylether and dried in a desiccator under vacuum. The product is thenpurified by flash chromatography using a dichloromethane/methanolmixture gradient from 90/10 to 70/30. Yield is 42%. The product hasabsorbance maximum in ethanol at 554 nm and emission maximum at 570 nm.

EXAMPLE 2 Sinthesis of 5-(cyanine IRIS 3 sulfo alkynyl)-2′-deoxycytidine(Compound 2)

15.6 mg (0.044 mmol) of 5-iodo-2′-deoxy-cytidine and 1,.7 mg (0.0088mmol) of CuI are placed in a vial with magnetic stirrer; 83.2 mg (0.131mmol) of Compound 1, dissolved in the minimal quantity of anhydrousN,N-dimethylformamide, are added. Argon is refluxed for fifteen minutes.0.012 mL (0.0088 mmol) of N(C₂H₅)₃ and 3.0 mg (0.0044 Mmol) of(C₆H₅)₂PdCl₂ are added and argon is refluxed fifteen minutes more. Thereaction is conducted for eight hours under stirring at roomtemperature. When the time has passed, 1.7 mg (0.0088 mmol) of CuI,0.012 mL of N(C₂H₅)₃ and 3.0 mg (0.0044 mmol) of (C₆H₅)2PdCl₂ are addedand the reaction is conducted in the same conditions 16 hours more.Purification is carried out on a MPLC column (eluents: CH₃OH, H₂O).

The product appears as a red coloured powdery solid.

EXAMPLE 3 Sinthesis of 5-(cyanine IRIS 3 sulfo alkynyl)-2′-deoxycytidinetriphosphate (Compound 3)

59 mg (0.071 mmol) of Compound 2, dissolved in the solvent (CH₃)₃PO₄,are placed in the reaction round-bottom flask, and argon is refluxed forthe whole length of the reaction. The solution is cooled at −10° C. and0.007 ml di POCl₃ are added. The solution is stirred at −10° C. forthirty minutes, 0.007 ml of POCl₃ are then further added and thetemperature is slowly raised to room temperature. After 100 minutes fromthe second POCl₃ addition monophosphorilation is complete. The reactionmixture is percolated into a solution of Na[NH(C₄H₇)₃]P₂O₇ inN,N-dimethylformamide pre-cooled at −10° C. After 100 minutes thesolution is added to N(C₂H₅)₃ (0.1 ml) dissolved in water (1.42 ml)pre-cooled at 0° C. The solution is stirred on ice for 15 minutes andlet to stay at 4° C. overnight. Purification is performed on reversephase MPLC.

EXAMPLE 4 Sinthesis of1-(4-carboxybutyl)-1′-es-5-ynil-3,3,3′,3′-tetrmethyl-5,5′-disulfonatedindodicarbocyanine sodium salt (Compound 4)

2.69 g (8.43 mmol) diofN-es-5-ynil-2,3,3-trimethylindoleninium-5-sulfonate, 4.84 g (16.98 mmol)of malonaldehyde dianil(N-[(1Z,2E)-3-anilinoprop-2-enylidene]benzenaminium chloride), 6.0 ml ofacetyl chloride and 60.0 mL of acetic anhydride are placed in 250 mlround-bottom flask. The mixture is heated at 120° C. for 90 minutes. Theorange solution is cooled to room temperature and added dropwise to 300ml of vigorous stirred diethyl ether. The product (hemicyanine) iscollected on a fritted glass filter, washed with diethyl ether and driedin a desiccator under vacuum. The desired product has an absorbancemaximum at 378 nm in methanol. Yield is 97%.

5.96 g (12.14 mmol) of the hemicyanine synthesized in the previous stepis placed in a 250 ml round-bottom flask together with 4,41 g (12,14mmol) of N-(4-carboxyibutyl)-2,3,3-trimethylindoleninium-5-sulfonatesodium salt, 10.80 ml of triethylamine and 109 ml of acetic anhydrideand heated at 135° C. for 2 h. The blue solution is cooled to roomtemperature and dropwise added to 800 ml of vigorously stirred diethylether. The desired product is collected on a fritted glass filter,washed with diethyl ether and dried in a desiccator under vacuum. Theproduct is then purified by flash chromatography using adichloromethane/methanol mixture gradient from 90/10 to 70/30. Yield is42%. The product has absorbance maximum in ethanol at 645 nm andemission maximum at 665 nm.

EXAMPLE 5 Synthesis of1-(4-carboxybutyl)-1′-{6-[N,N′-difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrril]-hex-5-ynil}-3,3,3′,3′-tetramethyl-5,5′-disulfonateindodicarbocyanine sodio salt (IRIS 5 alchinil-dimethylBDPY) (Compound5)

Compound 4 and N,N′-difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrinare reacted analogously to the procedure described in Example 2.

18.6 mg (0.044 mmol) diN,N′-difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin and 1.7 mg(0.0088 mmol) of CuI are placed in a vial with magnetic stirrer; 90.5 mg(0.131 mmol) of Compound 4 dissolved in the minimal quantity of dryN,N-dimethylformamide are added. Argon is refluxed for fifteen minutes.0.012 ml (0.0088 mmol) of N(C₂H₅)₃ and 3.0 mg (0.0044 mmol) of(C₆H₅)₂PdCl₂ are added and argon is refluxed for fifteen minutes more.The reaction is conducted for eight hours under stirring at roomtemperature. When the time has passed, 1.7 mg (0.0088 mmol) of CuI,0.012 mL of N(C₂H₅)₃ and 3.0 mg (0.0044 mmol) of (C₆H₅)₂PdCl₂ are addedand the reaction is conducted in the same conditions 16 hours more.Purification is carried out on a MPLC column (eluents: CH₃OH, H₂O). Thecompound of Formula 13 is obtained.

The product appears as a blue coloured powdery solid.

EXAMPLE 6 Labelling of DNA Through Reaction of Incorporation of5-(cyanine IRIS 3 sulfo alkynyl)-2′-deoxycytidine triphosphate by PCR

Nucleotides conjugates have been tested by PCR (Polymerase ChainReaction) to evaluate their efficiency as substrates for the polymerase.A plasmid derived from commercial vector pBluescript II SK (Statagene),in which has been cloned a fragment of cDNA derived from human genehGATA-3, has been used as DNA template. A fragment of the expectedlength of 1000 bp (base pairs) is generated using two standard T7(forward primer) and T3 (reverse primer) oligonuleotides as primers. PCRhas been conducted using several fluorescent alkynyl arm containingcyanine labelled nucleotide concentrations (from 0 to 50 μM), assummarized in the following table: Stock Final 10 20 30 50 Conc. Conc.C+ C− μM μM μM μM Template DNA 1 ng/μl 10 ng 10 μl — 10 μl 10 μl 10 μl10 μl 10× PCR buffer 10× 1× 5 μl 5 μl 5 μl 5 μl 5 μl 5 μl MgCl₂ 50 mM1.5 mM 1.5 μl 1.5 μl 1.5 μl 1.5 μl 1.5 μl 1.5 μl T7 primer (Fw) 10 μM0.5 μM 2.5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 μl T3 primer (Rv) 10 μM0.5 μM 2.5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 μl 2.5 μl d (ACT)P mix 3.3 mM 200μM 3 μl 3 μl 3 μl 3 μl 3 μl 3 μl dCTP 0.5 mM variable 20 μl 20 μl 19 μl18 μl 17 μl 15 μl 5-(cyanine IRIS 3 0.5 mM variable — — 1 μl 2 μl 3 μl 5μl alkynyl)-2′-dCTP Taq DNA Pol 2 U/μl 1 U 0.5 μl 0.5 μl 0.5 μl 0.5 μl0.5 μl 0.5 μl H₂O — to 5 μl 5 μl 15 μl 5 μl 5 μl 5 μl 5 μl

PCR reaction has been conducted according to the following instrumentalprotocol, using an iCycler (Biorad) thermocycler:

STEP 1(1 cycle)

-   -   94° C. 4 min

STEP 2 (10 cycles)

Denaturation 95° C. 1 min

“Annealing” 60° C. 1 min

After first cycle low temperature by 1.0° C. every cycle (“touch-down”method)

Extension 72° C. 1 min

STEP 3 (20 cycles)

Denaturation 95° C. 1 min

“Annealing” 50° C. 1 min

Extension 72° C. 1 min

STEP 4 (1 cycle)

-   -   4° C. infinite

After PCR, 1/10 of every reaction has been tested on 0.8% agarose gel.The remaining part has been purified by means of a QIAquick PCRpurification kit (Qiagen) according to the instrcons supplied. The DNAhas been then diluted in 120 μl of water and analyzed by means of aspectrophotometer and a fluorimeter verifying the typical absorbance andemission bands of the cyanine.

EXAMPLE 7 Conjugation of IRIS 5 alkynyl-dimethylBDPY with proteinkynasebioactive peptide

The proteinkynase inhibitor bioactive peptide (available throughSigma-Aldrich) used in this example has the sequence: Thr Thr Tyr AlaAsp Phe Ile Ala Ser Gly Arg Thr Gly Arg Arg Asn Ala Ile His Asp withfree terminal —NH₂ group.

29.55 mg di Compound 5 (IRIS 5 alkynyl-dimethyl-BDPY) (0.03 mmol), 12.36mg of Dicyclohexylcarbodiimide, 6.9 mg of N-hydroxysuccinimide and 3 mlof anhydrous N,N-dimethylformamide are placed in a 50 ml single neckround-bottom flask. The reaction is conducted at 70° C. for 5 hours.When the time has passed, a solution of the bioactive peptide (133.34 mgin 2 ml of DMF) is added to the reaction mixture and reacted at roomtemperature overnight. The desired conjugate is obtained which ispurified by medium pressure liquid chromatography (MPLC).

1. A cyanine modified with an alkynyl-linker arm, having the followinggeneral formula (I), including the valence tautomers thereof:

wherein R₁ is a linear, saturated or unsaturated alkyl chain, havingfrom 1 to 30 carbon atoms, wherein one or more carbon atoms are eachoptionally substituted by a component independently selected from anoxygen or a sulfur atoms, a —NH— or a —CONH— group, or a cyclic 4-, 5-or 6-membered grouping of carbon atoms, aromatic or not aromatic,wherein one or more carbon atoms are each optionally substituted by aheteroatom independently selected from oxygen, sulfur, nitrogen andselenium; WI and W₂ are independently selected from a benzene ring and anaphthalene ring wherein one or more carbon atoms are optionallysubstituted by one or more heteroatoms selected from oxygen, sulfur,selenium and nitrogen, or one of W₁ and W₂ is absent, or both of themare absent; X₁ and X₂ are independently selected from the groupconsisting of —O—, —S—, —Se—, —C(CH₃)₂, —NH— and —CH═CH—; and R₂, R₃,R₄, R₅ and R₆ are independently selected from hydrogen, —COOH, —OH,—NO₂, —OCH₃, —SO₃H, —SO₃—, and —R₈—Y wherein R₈ is a linear, saturatedor unsaturated alkyl chain, having from 1 to 30 carbon atoms, whereinone or more carbon atoms are each optionally substituted by a componentindependently selected by an oxygen or a sulfur atom, a —NH— or a —CONH—group, or a cyclic 4-, 5- or 6-membered grouping of carbon atoms,aromatic or not aromatic, wherein one or more carbon atoms are eachoptionally substituted by a heteroatom independently selected fromoxygen, sulfur, nitrogen or selenium, and wherein Y is selected from thegroup consisting of hydrogen, carboxyl, carbonyl, amino, sulphydryl,thiocyanate, isotyocianate, isocyanate, maleimide, hydroxyl,phosphoramidite, glycidyl, imidazolyl, carbamoyl, anhydride,bromoacetamido, chloroacetamido, iodoacetamido, sulphonyl halide, acylhalide, aryl halide, hydrazide, succinimidyl ester,hydroxysulfosuccinimidyl ester, phthalimidyl ester, naphthalimidylester, monochlorotriazine, dichlorotriazine, mono- or di-halidesubstituted pyridine, mono- or di-halide substituted diazine, aziridine,imidic ester, hydrazine, azidonitrophenyl, azide,3-(2-pyridyldithio)-propionamide, glyoxal, aldehyde, nitrophenyl,dinitrophenyl, trinitrophenyl and —C≡CH, provided that one of R₂, R₃,R₄, R₅ and R₆ is —R₈—Y, with Y being different from H and from —C≡CH; Mis a counterion; and Q is a polymethinic chain selected from:

wherein R₇ is selected from the group consisting of hydrogen, fluorine,chlorine, bromine, iodine, phenoxy, thiophenoxy, anilino,cyclohexylamino, piridine, —R₈—Y, —O—R₈—Y, —S—R₈—Y, —NH—R₈—Y, wherein R₈and Y are as defined above, and aryl optionally substituted by one ormore substituents independently selected from the group consisting of—SO₃H, carboxyl (—COOH), amino (—NH₂), carbonyl (—CHO), thiocyanate(—SCN), isothiocyanate (—CNS), epoxy and —COZ wherein Z represents aleaving group.
 2. The cyanine according to claim 1, wherein said leavinggroup is selected from the group consisting of —Cl; —Br; —I; —OH; —OR₁₁;—OCOR₁₁, wherein R₁₁ is linear or branched alkyl having from 1 to 4carbon atoms; —O—CO—Ar, wherein Ar is aryl optionally substituted;—O—CO—Het, wherein Het is selected from succinimide, sulfosuccinimide,phthalimide and naphthalimide; —NR₂₂R₃₃, wherein R₂₂ and R₃₃ are eachindependently linear or branched alkyl having from 1 to 10 carbon atoms.3. (canceled)
 4. The cyanine according to claim 2 selected from thegroup consisting of:

wherein Q and R₈ are as defined in claim 1 and n is an integer between 1and
 100. 5. The cyanine according to claim 1, conjugated through thelinker arm —R₁—C≡CH with a biomolecule.
 6. The cyanine according toclaim 5, wherein said biomolecule is selected from the group consistingof nucleotides, nucleosides, oligonucleotides, nucleic acids, peptidesand proteins.
 7. The cyanine according to claim 1, conjugated throughthe linker arm —R₁—C≡CH with a second fluorescent dye, said secondfluorescent dye being capable of emitting fluorescence at wavelengths atwhich the cyanine is capable of absorbing, or said fluorescent dye beingcapable of absorbing at wavelengths at which the cyanine is capable ofemitting.
 8. The conjugated cyanine according to claim 7, wherein saidsecond fluorescent dye isN,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin.
 9. Theconjugated cyanine according to claim 7, wherein said second fluorescentdye is a transition metal complex with at least one heterocyclicnitrogen-containing ligand.
 10. The cyanine according to claim 1,conjugated through the linker arm —R₁—C≡CH with a first biomoleculeselected from the group consisting of nucleotides, nucleosides,oligonucleotides, nucleic acids, peptides, proteins, vitamins andhormones, and through the linker arm —R₈—Y with a second equal ordifferent biomolecule selected from the group consisting of nucleotides,nucleosides, oligonucleotides, nucleic acids, peptides, proteins,vitamins and hormones. 11-12. (canceled)
 13. The use of a cyanineaccording to claim 1 as a fluorescent marker for biomolecules or as aquencher.